EP0576669A1 - Nouveaux procedes et compositions de traitement des maladies angiogeniques - Google Patents

Nouveaux procedes et compositions de traitement des maladies angiogeniques

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Publication number
EP0576669A1
EP0576669A1 EP19930904627 EP93904627A EP0576669A1 EP 0576669 A1 EP0576669 A1 EP 0576669A1 EP 19930904627 EP19930904627 EP 19930904627 EP 93904627 A EP93904627 A EP 93904627A EP 0576669 A1 EP0576669 A1 EP 0576669A1
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EP
European Patent Office
Prior art keywords
treatment
rpf4
synthetic
recombinant
medicament
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EP19930904627
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German (de)
English (en)
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EP0576669A4 (en
Inventor
Theodore E. Maione
Richard J. Sharpe
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Repligen Corp
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Repligen Corp
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Publication of EP0576669A1 publication Critical patent/EP0576669A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • C07K14/522—Alpha-chemokines, e.g. NAP-2, ENA-78, GRO-alpha/MGSA/NAP-3, GRO-beta/MIP-2alpha, GRO-gamma/MIP-2beta, IP-10, GCP-2, MIG, PBSF, PF-4, KC
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • Angiogenesis the development of new capillary blood vessels, is an important process in the developing fetus and growing human. However, in healthy adults, angiogenesis occurs significantly only during wound healing and in the menstrual cycle.
  • angiogenic dysfunctions include diabetic retinopathy, retrolental fibroplasia, neovascular glaucoma, psoriasis, angiofibromas, immune and non-immune inflammation (including rheumatoid arthritis) , capillary proliferation within atherosclerotic plaques, hemangiomas, and Kaposi's Sarcoma have also recently been recognized as diseases possessing characteristics of dysregulated endothelial cell division and capillary growth. These conditions along with growth of solid tumors are collectively referred to as "angiogenic diseases” (Folkman, J. , and M. Klagsbrun [1987] Science 235:442-447).
  • endothelial cell proliferation is pathological or, at least, unwanted.
  • endometriosis is characterized by the abnormal proliferation and positioning of certain endothelial cells which normally line the inner wall of the uterus. Control of the angiogenic process could help to prevent or alleviate endometriosis. Also, prevention of endothelial cell growth in the uterus could be a means of birth control.
  • Endothelial cell growth is associated with wound healing. This growth is undesirable during extended surgical proceedings and where excessive scar formation may occur. Therefore, a means of controlling endothelial cell proliferation would help prevent or reduce unwanted scar formation.
  • protamine which is a protein found only in sperm.
  • Protamine has been shown to inhibit tumor angiogenesis and subsequent tumor growth (Taylor, S. and J. Folkman [1982] Nature 297:307-312).
  • Protamine's anti-angiogenesis activity has been attributed to its well-known capacity to bind heparin (Taylor and Folkman [1982], supra) .
  • Clinical experiments with protamine have not been pursued because of the toxicity associated with protamine injection.
  • Protamine which is usually isolated from salmon sperm, is known to be antigenic in humans, and anaphylactic reactions to this protein have been observed with secondary exposures. At least two other compounds have been studied in regard to their heparin-binding activity: platelet factor 4 (PF4) and major basic protein. Major basic protein has demonstrated heparin-binding activity but is of little practical utility because of its high toxicity. Platelet factor 4 is a well-known protein which has been completely sequenced (Deuel, T.F., R.M. Senior, D. Chang, G.L. Griffin, R.L. Heinrikson, and E.T. Kaiser [1981] Proc. Natl. Acad. Sci. USA 78:4585-4587).
  • PF4 In addition to angiostatic properties, PF4 possesses characteristic structural features of the pro- inflammatory proteins interleukin-8 and ⁇ -thromboglobulin and has been shown to be chemotactic for neutrophils and monocytes in vivo (Wolpe and Cera i [1989] the FASEB Journal, 3:2565-2573). This similarity of the structure and activities of PF4 to well characterized pro- inflammatory proteins along with the ubiquitous aggregation of platelets at sites of inflammation suggest that PF4 may be an endogenous mediator of inflammation. Thus, it is anticipated that swelling could accompany the administration of PF4 in vivo. There is a significant and very long-standing need to locate an effective and non-toxic inhibitor of angiogenesis and endothelial cell proliferation. Angiogenesis plays a major role in the initiation and progression of widespread catastrophic illnesses, including cancer. An effective, non-toxic agent which can be administered locally and/or systemically to treat these illnesses would be highly advantageous and has long eluded identification.
  • conservative amino acid substitution means the substitution of an amino acid with another amino acid that is biologically compatible with the first amino acid.
  • PF4 has clinical utility in the treatment of diseases which involve angiogenesis and endothelial cell proliferation. Furthermore, PF4 fragments are demonstrated to be inhibitors of angiogenesis. The ability to inhibit angiogenesis has been found in synthetic peptides corresponding to sequences in PF4 as small as the carboxyterminal 13 amino acids.
  • the invention features treatment of angiogenic diseases with a combination of PF4 and an anti- inflammatory agent.
  • Anti-inflammatory agents help to alleviate unwanted swelling, pain, or tissue damage which could accompany the administration of pro-inflammatory compounds.
  • the invention also features methods for the treatment of tumors that contain malignant endothelial cells with PF4 either alone or in combination with an anti-inflammatory agent.
  • the invention features methods for the treatment of brain tumors with PF4.
  • Figure 1 shows DNA and amino acid sequence of native rPF4.
  • Figure 2 shows the inhibition of angiogenesis resulting from the treatment of rPF4 and various related peptides.
  • Figure 3 depicts the inhibition of endothelial cell proliferation by rPF .
  • Figure 4 depicts the alpha-helical configurations of rPF4 and rPF4-241.
  • Figure 5 compares the inhibition of angiogenesis resulting from treatment with rPF4 and rPF4-241.
  • Figure 6 compares the inhibition of human umbilical vein endothelial cell proliferation resulting from treatment with rPF4 or rPF4-241.
  • Figure 7 shows the ability of rPF4 and rPF4-241 to inhibit tumor growth.
  • Figure 8 shows footpad swelling in mice as a function of time after injection with either rPF4, rPF4 and indomethacin, or a buffer solution.
  • Figure 9 shows quantification of inflammatory cell infiltrate after treatment with rPF4 or rPF4 with indomethacin.
  • Figure 10 shows tumor growth after administration of rPF4 alone, indomethacin alone, buffer alone, or rPF4 and indomethacin.
  • the subject invention pertains to in vivo inhibition of angiogenesis by rPF4 and certain analogs and peptide fragments of PF4. These analogs and peptide fragments of PF4 can be used to treat angiogenic diseases.
  • angiogenic disease refers to growth of solid tumors, and other conditions involving angiogenic dysfunctions including macular degeneration.
  • the subject invention also concerns the use of rPF4 and PF4 fragments for treatment of diseases of dysregulated endothelial cell proliferation.
  • the types of solid tumors that can be treated by rPF4, PF4, or analogs and fragments thereof, include all types of lung tumors, including small cell lung carcinoma, tumors of the breast, colon/rectum, prostate, head and neck, stomach, bladder, kidney, pancreas, liver, ovary and uterus; sarcomas; melanoma and other metastatic skin cancers; nonmetastatic skin cancers (e.g., Kaposi's sarcoma, basal cell carcinoma) ; and, most preferably tumors of the brain.
  • the types of tumors that can treated using the compositions and methods of the invention include tumors that are not surgically accessible and/or are resistant to chemotherapy and radiation therapy.
  • Advanced invasive malignancy with or without surgery, can be treated using the compositions and methods of the invention.
  • the invention can be used as an adjuvant therapy following surgical resection and can be used to treat known metastatic disease and nonmetastatic cancer.
  • Treatment of the tumors and diseases described above can be either systemic, regional, or local (intralesional) , depending upon the type and severity of the disease as well as the accessibility of the disease site.
  • Systemic treatment includes intravenous bolus injections and infusions, subcutaneous injections, implants, refillable reservoirs and sustained release depots and intramuscular injections.
  • Regional treatment includes intraarterial for the treatment of primary liver tumors and liver metastases, and for the treatment of kidney, brain and pancreatic tumors.
  • Regional intraperitoneal treatment can be used for the treatment of tumors of the ovary.
  • Local treatment can be used for tumors of the brain, uterus, bladder, head and neck, for Kaposi's sarcoma and other nonmetastatic skin cancers, for metastatic skin cancer once dissemination precludes further surgical excision, and for colon and rectal cancer.
  • Brain tumors are generally treated with initial surgical excision if possible, followed by intensive chemotherapy and radiation therapy.
  • Aggressive brain tumors (high grade astrocytoma, glioblastoma multiforme) ultimately produce mortality by reoccurrence near their original site due to incomplete surgical removal, rather than through metastatic dissemination.
  • Methods to specifically focus postsurgical treatment on the site of the initial lesion are therefore desirable to deliver effective therapy and reduce damage to healthy tissues.
  • the subject invention in part takes advantage of the ability of rPF4 to inhibit capillary formation in vivo as well as embryonic neovascularization. Full length recombinant PF4 also inhibits growth factor- dependent human endothelial cell proliferation in vitro.
  • PF4 directly inhibits growth of pure cultures of endothelial cells indicates that, advantageously, its effects are not mediated by some other cell type.
  • the activity of the C-13 peptide is especially surprising in light of its inability to affect the anticoagulant activity of heparin.
  • the use of the C-13 peptide offers several advantages over whole rPF4 such as reduced dosage (weight basis) , reduced likelihood of antigenicity, and greater likelihood of effectiveness in novel dosage forms.
  • the C-13 peptide of PF4 also retains the ability to prevent Con-A induced immunosuppression in mice, an activity which is unaffected by heparin and probably independent of the ability of the peptide to inhibit angiogenesis.
  • angiogenesis is required for solid tumors to grow beyond a few cubic millimeters.
  • use of rPF4, or a fragment thereof, to inhibit tumor growth by inhibiting angiogenesis presents a novel and highly advantageous means of therapy, although efficacy of PF4 in some of the therapies described herein is not entirely explained by inhibition of angiogenesis; for example, we have discovered that in specific types of cancers that contain malignant endothelial cells, such as Kaposi's sarcoma, PF4 was inhibitory.
  • the C-13 peptide inhibits angiogenesis without affecting the anticoagulant activity of heparin demonstrates that this small peptide would also have the benefit of not interfering with concurrent anticoagulant therapy.
  • small peptides are generally less antigenic than larger proteins, and, thus, the PF4 fragments can be used advantageously for oral and transdermal administration. These types of delivery are particularly useful in the treatment of gastrointestinal capillary proliferation (e.g., Kaposi's Sarcoma) and skin lesions, respectively. Intralesional, as well as systemic, administration of PF4 fragments are also appropriate for treatment of these conditions.
  • rPF4-241 Analogs of PF4 were created which lack heparin binding activity but retain ability to inhibit angiogenesis.
  • One such analog known as rPF4-241
  • rPF4-241 was created by cassette mutagenesis of a synthetic PF4 gene whereby the DNA sequence encoding the four lysine residues near the carboxy terminus of PF4 were converted to a sequence encoding two Gln-Glu couplets.
  • rPF4-241 is administered intralesionally, it can be applied such that the dosage is between about 1 ⁇ qflesion and about 4 mg/lesion.
  • dosages of rPF4 and fragments thereof may be twice that of rPF4-241 or higher.
  • PF4 has been shown to be chemotactic for neutrophils and monocytes m vitro, suggesting that it may mediate an inflammatory response.
  • rPF4 recombinant human PF4
  • Injection of an equivalent amount of cytochrome C, buffer alone, or an amino terminal PF4 peptide failed to elicit a significant inflammatory response, however, the carboxy terminal PF4 peptide was pro-inflammatory.
  • the inflammatory infiltrate induced by both rPF4 and the 41 amino acid COOH terminal peptide was composed of neutrophils and to a lesser degree mononuclear cells-. Although relatively high concentrations of rPF4 are required to elicit an inflammatory response, these concentrations may be locally obtainable during platelet aggregation or at sites of administration of rPF4 or related compounds.
  • the rPF4 pro- inflammatory effect was significantly suppressed by systemic administration of an anti-inflammatory agent without reducing the angiostatic activity.
  • CAM Chicken Chorioallantoic Membrane Assay. Fertile eggs were incubated in a stationary position for 3 days at 37°C and 70-80% relative humidity. During this time, the embryo rose to the upper surface of the egg contents. At the beginning of the 4th day, the eggs were cracked without inversion and carefully deposited into sterile plastic petri dishes such that the embryo remained on the upper surface. The shell-free eggs were incubated for an additional 72 hours at 37°C, under an atmosphere containing 2.5-3.5% C0 2 after which the growing embryos developed a recognizable CAM. Discs, made by mixing test samples with 1% (w/v) methylcellulose, were dried and placed on the CAM between major veins and approximately 0.5 cm from the embryo.
  • the cultures were harvested by trypsin treatment, diluted, replated, and grown to confluence. Prior to the start of an experiment, the cells were centrifuged and resuspended in heparin-free media and incubated with the test substance (PF4) for 3 days under standard culture conditions. At the end of the incubation period, the cells were removed by trypsin treatment and counted with a Particle Data Elzone 180 Cell Counter. Statistical significance between means was determined by a standard Student t-test for unpaired data.
  • Inhibition of DNA synthesis was measured by plating the cells as described, then incubating with the test substance for 24 hours. 3 H-Thymidine (1 ⁇ ,Ci/well) was added for an additional 6 hours and the plates were frozen at —70 C. Following 2 freeze/thaw cycles, the cells were aspirated onto a fiber filter, washed with distilled water, fixed with MeOH, and counted for incorporation of radioactivity into DNA.
  • rPF4 in preventing in vivo tumor growth and angiogenesis
  • tumor bearing animals were injected daily, directly into the nascent tumor, with either rPF4 or with buffer lacking rPF4, beginning one day after tumor inoculation.
  • Tumor volume was measured at regular intervals with digital calipers by laboratory personnel uninformed of the specific treatment received by each subject animal.
  • Footpad Assay 0.05 ml of PBS containing a test substance was injected intradermally into the right hind footpad of each mouse. An identical amount of diluent, not containing the test substance, was injected into the left hind footpad. At various time points, footpad thicknesses were measured with a spring loaded engineer's micrometer (Fowler Co., Biggswald, England).
  • mice were sacrificed and footpad tissue was prepared for light microscopy. This tissue was used to quantify infiltrating cell types. Biopsy specimens were fixed in 10% buffered formalin for at least 48 hours and then prepared using standard techniques of paraffin embedding and staining with hematoxylin and eosin. Using an ocular grid, four cellular areas of dermis in each specimen were examined in a coded fashion at 1000X magnification and inflammatory cells were quantified. Differences between groups were assessed by Student's t test or analysis of variance, where appropriate. rPF4 Production.
  • Recombinant PF4 was produced in F ⁇ coli as an N-terminal fusion protein containing a unique methionine residue immediately preceding the PF4 portion. More specifically, expression plasmid pPF4-211 was constructed by cloning a synthetic gene encoding native sequence PF4 (Figure 1) (Poncz et al. [1987] Blood 69:219) into the multiple restriction site region of plasmid pREV2.2 (deposited July 30, 1986; accession # NRRL B-18091) . Codon usage in the synthetic gene was optimized for expression in E . coli. and synthetic DNA linkers were included on each end to facilitate the directional insertion of the PF4 gene into the vector.
  • Cells expressing the fusion protein were subjected to lysozyme (1 mg/g cells) , DNase I (500 units/100 g cells) and bead mill treatments.
  • the lysis pellet containing the fusion protein was treated with CNBr (10 g/lOOg cells) in 70% formic acid to cleave the fusion protein at the methionine between the BG and PF4 portions.
  • CNBr 10 g/lOOg cells
  • the recombinant protein was extracted with 200 ml of 50 mM Tris-Cl, pH 7.6, 5 mM EDTA, and 10 mM DTT per 100 g of cell starting material.
  • Native sequence rPF4-211 was purified by binding the protein to heparin agarose, removing contaminating proteins with 0.6 M NaCl, and eluting with 1.2 M NaCl. The resulting material was dialyzed into 20 mM sodium acetate, pH 4.0, and analyzed on a 15% SDS-PA gel stained with Coomassie Brilliant Blue. Minor contaminants could be removed using C 4 reverse phase high pressure liquid chromatography (HPLC) to prepare the protein for in vivo use.
  • HPLC high pressure liquid chromatography
  • rPF4-241 Production of rPF4-241 and other PF4 analogs.
  • a synthetic gene encoding the mutant designated rPF4-241 was constructed by changing the codons for the four lysine residues near the C-terminus of PF4 to sequences encoding two Gln-Glu couplets (CAA GAA) by cassette mutagenesis between the Bbel and Smal sites. Linkers were included at the ends of the synthetic gene, and the gene was inserted into pREV2.2 as described above. Genes encoding other PF4 mutants or analogs were prepared in a similar manner.
  • mutant proteins e.g., rPF4-241
  • the mutant proteins were cleaved and extracted as described above.
  • the extracts were then purified using DEAE-Sepharose chromatography, and eluted with a gradient of 0-1 M NaCl.
  • the PF4 proteins generally eluted at approximately 0.5 M NaCl and were dialyzed into 20 mM phosphate buffer, pH 7.5.
  • the samples were further purified by reverse phase HPLC.
  • PF4 peptides Peptides were prepared by standard solid phase synthesis procedures, cleaved from the solid support and deblocked, and purified by reverse phase HPLC. Reagents. Recombinant human IL-1 (rIL-1) was purchased from Genzyme Corporation (Cambridge, MA) . Cytochrome c and J coli endotoxin were purchased from Sigma Chemical Co. (St. Louis, MO) . Slow release indomethacin pellets were purchased from Innovative Research (Toledo, OH) .
  • mice C57B1/6J, A/J and C3H/HeJ female mice, 6-8 weeks old, were purchased from the Jackson Laboratory (Bar Harbor, ME) . Following are examples which illustrate procedures, including the best mode, for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.
  • the lysine rich region of PF4 (residues 61-66) is also the domain associated with the binding of heparin by PF4.
  • Heparin is known to play a role in modulating angiogenesis, which can also be affected by protamine, another well characterized heparin-binding protein.
  • protamine another well characterized heparin-binding protein.
  • PF4-based synthetic peptides To assess the ability of PF4-based synthetic peptides to bind heparin, we assayed the activity of coagulation- cascade enzymes which are inhibited by heparin.
  • the Factor Xa assay used here has previously been described in Denton et al. (1983) Biochem. J. 209:455-460.
  • Protamine and platelet factor 4 are able to prevent the heparin inhibition of thrombin and Factor Xa at approximately equimolar concentrations.
  • the 41 amino acid C-terminal peptide of PF4 (C-41) prevented heparin inhibition less effectively, but the C-13 peptide was unable to prevent the inhibition of thrombin even at concentrations ten times that of an effective level of rPF4. This unexpected finding suggests that the C-13 peptide inhibits angiogenesis by some method other than heparin binding.
  • rPF4 having the wild type sequence (rPF4-211) and related peptides to inhibit growth factor-stimulated human endothelial cell proliferation in vitro. As shown in Figure 3, rPF4 significantly inhibited endothelial cell growth in a dose-dependent fashion at a concentration as low as 1.3 ⁇ -M. Inhibition was complete at 3.2 ⁇ M in the heparin- deficient medium employed here.
  • Example 5 Construction of rPF4-2 1 15 A mutant of PF4 was created by converting the four lysine residues at the carboxy terminus of PF4 to two Gln-Glu couplets as disclosed above. This protein apparently retains the alpha-helical secondary structure ( Figure 4) for this region of the molecule with the 20 concurrent loss of heparin binding activity.
  • the protein was reactive with polyclonal antibodies to native PF4 and was determined to possess the appropriate modifications by amino acid analysis. Significantly, the purified mutant protein lacked 25 heparin-binding activity in the Factor Xa inhibition assay.
  • C-13-241 has the following 30 sequence:
  • rPF4-241 was tested for its ability to 35 inhibit capillary growth in the chicken chorioallantoic membrane (CAM) assay. Even at the lowest concentrations tested (1.25 nmol/disc) rPF4-241 extensively inhibited angiogenesis in the CAM system ( Figure 5) . This inhibition was even more effective than that caused by equal concentrations of native rPF4 as suggested by larger avascular zones on the membrane. The inhibitory effect of rPF4-241 was not reversed by heparin.
  • CAM chicken chorioallantoic membrane
  • Example 8 Inhibition of In Vivo Tumor Growth
  • the efficacy of rPF4-211 or rPF4-241 in preventing tumor growth and angiogenesis was tested.
  • the inhibition of in vivo tumor growth was assayed after injection of either rPF4-211 (in 20 mM NaOAc, pH 4.0) or rPF4-241 (in 50 mM sodium phosphate, pH 6.5, 50 mM NaCl) directly into the nascent tumor, as described in the materials and methods section above.
  • rPF4-211 in 50 mM sodium phosphate, pH 6.5, 50 mM NaCl
  • rPF4 as an inhibitor of angiogenesis, will possess clinical usefulness in the management of malignant melanoma and other cancers. Progressive growth of tumors requires new blood vessel formation which, if inhibited, may not only restrict tumor growth, but stimulate regression of . existing vessels, as well as enhance other responses to malignant invasion.
  • the finding that rPF4 inhibition of in vivo tumor growth was apparent within three days of the initial inoculation (of rPF4) indicates that rPF4 acts to modulate tumor growth by local mechanisms rather than by immunomodulation which would require a longer time course. Additionally, rPF4 did not directly inhibit tumor cell growth in vitro. It appears, therefore, that rPF4 modulated the host's angiogenic response to the growing tumor.
  • Example 9 It has been shown that proteins of identified structure and function may be constructed by changing the amino acid sequence if such changes do not significantly alter the protein secondary structure (Kaiser, E.T., and F.J. Kezdy [1984] Science 223:249-255)-.
  • the subject inventionr includes other mutants or fragments of the PF4 sequences depicted herein which lack affinity for heparin and exhibit substantially the same or higher angiostatic activity.
  • a preferred region for modification is the lysine rich region near the carboxy terminus corresponding to the heparin binding domain (residues 60- 70) .
  • amino acids 60 through 70 cannot be eliminated.
  • the subject invention includes mutants of the amino acid sequences depicted herein which do not alter the protein secondary structure, or if the structure is altered, the biological activity is retained.
  • conservative substitutions of amino acids may be made.
  • amino acids may be placed in the following classes: basic, hydrophobic, acidic, polar, and amide. Substitutions whereby an amino acid of one class is replaced with another amino acid of the same type fall within the scope of the subject invention so long as the substitution does not materially alter the biological activity of the compound.
  • Table 2 provides a listing of examples of amino acids belonging to each class.
  • non-conservative ' substitutions can also be made.
  • a lysine residue near the C-terminus of PF4 may be replaced with any of the following amino acids: E, Q, D, N, M, A, L, and I.
  • the critical factor is that these substitutions must not significantly detract from the biological activity of the rPF4 or the rPF4 fragment.
  • rPF4-302 which does not exhibit significant activity in either the CAM or the HUVEC assay, has no charged amino acid residues between residues 60 and 70.
  • rPF4-231 which also does not exhibit significant biological activity, terminates at amino acid number 60. If a person skilled in the art wished to investigate the biological activity of other rPF4 mutants, it would now be a straightforward procedure to make the desired mutations and test the resulting peptides for activity. Using the teachings of this document, the researcher could prepare and readily test peptides which could be expected to have the desired properties. For example, the amino acid substitutions just described for the full length rPF4 molecule can also be made with the C-13 and C-41 fragments which are described above.
  • Example 10 Inflammatory Properties of rPF4 and Related Compounds
  • the time course of rPF4 induced inflammation shows a rapid increase from baseline and peaks at between 6 and 12 hours and almost completely resolves by 36 hours.
  • Example 11 Effects of Anti-Inflammatory Agent with rPF4
  • 0.05 mg, slow release indomethacin pellets (Innovative Research, Toledo, OH) were implanted subcutaneously under light ether anesthesia 48 hours prior to an experiment. These pellets continuously release their contents over 14 days.
  • indomethacin can be used to decrease the swelling which could accompany the administration of PF4 or PF4-related substances.
  • Other non-steroidal anti- inflammatory agents could also be used.
  • the anti- inflammatory agents useful in the combinations and methods of this invention include steroidal and non- steroidal anti-inflammatory agents.
  • the non-steroidal anti-inflammatory agents include, but are not limited to, acetyl salicylic acid (aspirin) , methyl salicylate, sodium salicylate, phenylbutazone, oxyphenbutazone, apazone, indomethacin, sulindac, tol etin, mefenamic acid, ibuprofen, naproxen, fenoprofen, flurbiprofen, ketoprofen, and other compounds.
  • Other anti-inflammatory agents useful in the combinations and methods of this invention are lipocortins derived from natural sources or lipocortins and lipocortin-like polypeptides produced by recombinant techniques (see United States patent applications Serial Nos.
  • Steroidal anti-inflammatory agents which could be used according to the subject invention include, but are not limited to, hydrocortisones.
  • Example 12 Anti-Tumor Activity of rPF4 Combined with Indomethacin
  • mice Four groups of mice were used in this experiment. In two groups of mice, slow release indomethacin pellets (50 ⁇ g) were implanted surgically under the skin of the left flank. The other two groups were not treated with indomethacin. Tumors were implanted subcutaneously in all four groups in the right flank.
  • the combinations and methods of the present invention may allow the administration of PF4, or related compounds, in higher doses in some cases than those tolerated in conventional treatment regimes based upon PF4 alone. Accordingly, the combinations and methods of this invention advantageously reduce or eliminate the inflammatory effects of high dose treatments with PF4 alone. Thus, the use of PF4 in combination with an anti- inflammatory agent may reduce the duration of treatment which would be required by therapies based upon conventionally tolerated lower dosages of PF4 alone.
  • the combinations and methods of this invention are useful in treating any mammal, including humans.
  • mammals are treated with pharmaceutically effective amounts of the two active components—PF4 and an anti-inflammatory agent—of the combinations of this invention for a period of time sufficient to inhibit angiogenesis or endothelial cell proliferation.
  • PF4 an anti- inflammatory agent and the PF4 (or PF4-related compounds) are administered sequentially or concurrently to the patient.
  • the most effective mode of administration and dosage regimen of PF4 and anti-inflammatory agent will depend upon the type of disease to be treated, the severity and course of that disease, previous therapy, the patient's health status, and response to PF4 and the judgment of the treating physician.
  • PF4 may be administered to the patient at one time or over a series of treatments.
  • the anti-inflammatory agent and the PF4 are administered sequentially to the patient, with the anti-inflammatory agent being administered before, after, or both before and after treatment with PF4.
  • Sequential administration involves treatment with the anti-inflammatory agent at least on the same day (within 24 hours) of treatment with PF4 and may involve continued treatment with the anti-inflammatory agent on days that the PF4 is not administered.
  • Conventional modes of administration and standard dosage regimens of anti- inflammatory agents may be used (see Gil an, A.G. et al. [eds.] The Pharmacological Basis of Therapeutics, pp. 697-713, 1482, 1489-91 [1980]; Physicians Desk Reference, 1986 Edition) .
  • indomethacin may be administered orally at a dosage of about 25-50 mg, three times a day. Higher doses may also be used.
  • aspirin about 1500-2000 mg/day
  • ibuprofen about 1200-3200 mg/day
  • conventional therapeutic doses of other anti-inflammatory agents may be used. Dosages of anti-inflammatory agents may be titrated to the individual patient.
  • the patient may receive concurrent treatments with the anti- inflammatory agent and PF4.
  • Local, intralesional, or intravenous injection of PF4 is preferred (see Gilman et al. , supra at pp. 1290-91) .
  • the anti-inflammatory agent should preferably be administered by subcutaneous injection, subcutaneous slow-release implant, or orally.
  • the patient may receive a composition comprising a combination of PF4 (or PF4- related compounds) and an anti-inflammatory agent according to conventional modes of administration of agents which exhibit anticancer, antitumor, or anti- inflammatory activity. These include, for example, parenteral, subcutaneous, intravenous, or intralesional routes of administration.
  • Example 14 Dosages for Systemic Administration It has been discovered that very high doses of PF4 are required when PF4 is used systemically for treatment of tumors, and that these high doses do not have unacceptable high levels of toxicity. An experiment demonstrating the need for, and tolerance of, high doses of PF4 was carried out as follows.
  • PF4 produced a dose-dependent effect, as measured by both parameters. Optimal results were observed when PF4 was administered systemically, when dosages in excess of 5000 ⁇ g per kg of body weight; at these dosages, no observable toxicity or other adverse effects were noted.
  • Example 17 Systemic Treatment.
  • PF4 can be administered by direct intravenous injection, or preferably by intravenous infusion lasting from 0.5 to 4 hours per single treatment.
  • Patients can be treated as in- or out-patients. Patients may also be treated using implantable subcutaneous portals, reservoirs, or pumps. Multiple intravenous or subcutaneous doses are possible, and in the case of implantable methods for treatment, formulations designed for sustained release will be especially useful.
  • Patients can be treated at dosages of 0.3 to 12 g of rPF4 per period; preferably with 4 to 180 mg/kg in a volume of 60 ml to 2.5 liters per day.
  • a dosage is defined as a single dose administered as a bolus injection or intravenous infusion; or the compound can be administered to the patient as an intravenous infusion over a period of a day; alternatively, the compound can be administered in several bolus injections interrupted by periods of time such that the dose is delivered over the course of a 24 hour period.
  • the most preferred method of treatement is to administer the compound to the patient in one injection or infusion per day.
  • Patients may be treated daily on alternative weeks for six weeks, or possibly for life. They may also be treated three times per week continuously, or they may be treated daily for life.
  • Regional treatment is useful for treatment of cancers in specific organs in the patient, including, but not limited to primary liver cancer, brain and kidney cancer and liver metastases from colon/rectal cancer.
  • Treatment can be accomplished by intraarterial infusion.
  • a catheter can be surgically or angiographically implanted to direct treatment to the affected organ.
  • a subcutaneous portal, connected to the catheter can be used for chronic treatment, or an implantable, refillable pump may also be employed.
  • Patients can receive 0.05 to 1 g rPF4 (1 to 20 mg/kg) in a volume of 10 to 400 ml per single dose.
  • the schedule for treatment is the same as that described above for systemic treatment.
  • compositions used in these therapies may also be in a variety of forms. These include, for example, solid, semi-solid, and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspension, liposomes, suppositories, injectable and infusible solutions. The preferred form depends on the intended mode of administration and therapeutic application.
  • the compositions also preferably include conventional pharmaceutically acceptable carriers and adjuvants which are known to those of skill in the art.
  • the compositions of the invention are in the form of a unit dose and will usually be administered to the patient one or more times a day.
  • PF4 or related compounds may be administered to the patient in any pharmaceutically acceptable dosage form, including intravenous, intramuscular, intralesional, or subcutaneous injection.
  • An effective dose may be in the range of from about 0.003 to about 200 mg/kg body weight, it being recognized that lower and higher doses may also be useful. As discussed above, very high doses are preferred for systemic administration. It should, of course, be understood that the compositions and methods of this invention may be used in combination with other therapies.
  • a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained. When the symptoms have been alleviated to the desired level, treatment should cease. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.

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Abstract

Compositions et procédés de traitement de l'angiogénèse à l'aide du facteur plaquettaire 4 et de ses fragments peptidiques.
EP93904627A 1992-01-16 1993-01-19 Novel methods and compositions for treatment of angiogenic diseases Withdrawn EP0576669A4 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US82237892A 1992-01-16 1992-01-16
US822378 1992-01-16

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EP0576669A1 true EP0576669A1 (fr) 1994-01-05
EP0576669A4 EP0576669A4 (en) 1996-05-08

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EP (1) EP0576669A4 (fr)
JP (1) JPH06506702A (fr)
AU (1) AU3592593A (fr)
CA (1) CA2106368A1 (fr)
WO (1) WO1993013794A1 (fr)

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BE1006437A3 (fr) * 1992-12-10 1994-08-30 Company S A Z Sequence nucleotidique destinee au traitement du cancer et des infections.
US5348942A (en) * 1993-03-12 1994-09-20 Xoma Corporation Therapeutic uses of bactericidal/permeability increasing protein products
CN1105574C (zh) * 1993-03-12 2003-04-16 爱克索马技术有限公司 杀菌/增强通透性蛋白质产物的治疗用途
US5652332A (en) * 1993-03-12 1997-07-29 Xoma Biologically active peptides from functional domains of bactericidal/permeability-increasing protein and uses thereof
US5733872A (en) * 1993-03-12 1998-03-31 Xoma Corporation Biologically active peptides from functional domains of bactericidal/permeability-increasing protein and uses thereof
BR9607427A (pt) * 1995-02-16 1998-06-23 Hoffmann La Roche Inibiçao de angiogêne usando interleucina 12
FI955489A0 (fi) * 1995-11-15 1995-11-15 Antti Aarne Ilmari Lange Foerfarande foer adaptiv Kalmanfiltrering i dynamiska system
EP2287183A1 (fr) * 2009-07-29 2011-02-23 Institut National De La Sante Et De La Recherche Medicale (Inserm) Mutants PF4 polypeptides exhibiting an increased anti-angiogenic activity

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WO1985004397A1 (fr) * 1984-03-23 1985-10-10 Oncogen Regulateur de croissance apparente aux thrombocytes
EP0324556A2 (fr) * 1988-01-14 1989-07-19 Nippon Mining Company Méthode d'amélioration de la translation de mRNA et son application pour la production du facteur de trombocyte-4
EP0378364A2 (fr) * 1989-01-10 1990-07-18 Repligen Corporation Analogues de PF4 et leurs fragments et compositions pharmaceutiques les contenant
EP0407122A1 (fr) * 1989-07-06 1991-01-09 Repligen Corporation Compositions de PF4 modifié et méthode d'utilisation
WO1992002240A2 (fr) * 1990-07-27 1992-02-20 Repligen Corporation Compositions et procedes nouveaux pour le traitement des maladies angiogeniques
WO1993002192A1 (fr) * 1991-07-15 1993-02-04 Repligen Corporation Compositions pf4 et procedes d'utilisation desdites compositions

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AU623589B2 (en) * 1987-03-02 1992-05-21 Bristol-Myers Squibb Company Platelet related growth regulator

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Publication number Priority date Publication date Assignee Title
WO1985004397A1 (fr) * 1984-03-23 1985-10-10 Oncogen Regulateur de croissance apparente aux thrombocytes
EP0324556A2 (fr) * 1988-01-14 1989-07-19 Nippon Mining Company Méthode d'amélioration de la translation de mRNA et son application pour la production du facteur de trombocyte-4
EP0378364A2 (fr) * 1989-01-10 1990-07-18 Repligen Corporation Analogues de PF4 et leurs fragments et compositions pharmaceutiques les contenant
EP0407122A1 (fr) * 1989-07-06 1991-01-09 Repligen Corporation Compositions de PF4 modifié et méthode d'utilisation
WO1992002240A2 (fr) * 1990-07-27 1992-02-20 Repligen Corporation Compositions et procedes nouveaux pour le traitement des maladies angiogeniques
WO1993002192A1 (fr) * 1991-07-15 1993-02-04 Repligen Corporation Compositions pf4 et procedes d'utilisation desdites compositions

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Title
See also references of WO9313794A1 *

Also Published As

Publication number Publication date
JPH06506702A (ja) 1994-07-28
EP0576669A4 (en) 1996-05-08
AU3592593A (en) 1993-08-03
WO1993013794A1 (fr) 1993-07-22
CA2106368A1 (fr) 1993-07-17

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